The encapsulated fungus Cryptococcus neoformans is responsible for life-threatening disease, particularly in the context of compromised immunity, and current therapy is not adequate. The main virulence factor of C. neoformans is an extensive polysaccharide capsule. The structures of the capsule polysaccharides are known and aspects of capsule construction have been studied, but there is only limited understanding of how the events of capsule synthesis are regulated. Our long-term goal is to understand capsule synthesis in detail, so that we can target this process for antifungal therapy. In this application we propose to use a powerful combination of molecular and computational techniques to reconstruct the regulatory network that controls capsule synthesis. Aim I of the current application is designed to systematically identify genes involved in capsule regulation. Aim II is designed to computationally reconstruct the capsule regulatory network, enabling quantitative modeling of events in capsule regulation and prioritization of interactions for further study. Aim III proposes to validate selected regulatory relationships by modulating the expression levels of key transcription factors and to characterize the binding sites of these transcription factors. Methods will include gene deletion, gene expression analysis, microscopy, automated network reconstruction, quantitative modeling, RNA interference, tetracycline regulation, and chromatin immunoprecipitation coupled with sequencing. This range of studies will be enabled by the synergistic efforts of two labs with complementary skill sets, a potent combination that will generate significant progress in this important research field. Implementation of this innovative combination of techniques will yield greater understanding of cryptococcal biology and pathogenesis, identify targets for anti-fungal drug discovery, and generate valuable data sets and experimental approaches for future work on this and other eukaryotic pathogens. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because the organism under study causes serious human illness for which current therapies are not adequate. The capsule that surrounds Cryptococcus neoformans is essential to its ability to cause disease, and understanding how this structure is regulated may advance treatment of cryptococcal infection. This work will increase our understanding of fundamental processes in this pathogen and expand basic science knowledge about gene regulation, which will in turn contribute insights into the biology of eukaryotic organisms in general.